Ghrelin Protects Against Ethanol-Induced Gastric Ulcers in Rats: Studies on the Mechanisms of Action
Abstractq/#r*, 百拇医药
Ghrelin, the endogenous ligand for GH secretagogue receptors, has been reported to influence acid gastric secretion and motility, but its potential gastroprotective effect is unknown. The aims of this study were 1) to examine the effects of central and peripheral administration of ghrelin on ethanol-induced gastric ulcers in conscious rats, and 2) to investigate the possible roles of nitric oxide (NO), vagal nerve, and sensory fibers in the gastric effects of ghrelin.q/#r*, 百拇医药
Ghrelin was administered either intracerebroventricularly or sc 30 min before ethanol, and mucosal lesions were examined macroscopically. Additionally, rats were either treated with the inhibitor of NO synthesis N{omega} -nitro-L-arginine methyl ester (L-NAME) or underwent bilateral cervical vagotomy or capsaicin-induced sensory denervation. Conventional histology and immunohistochemistry for ghrelin, gastrin, and somatostatin were performed on gastric specimens from representative rats.
Central ghrelin (4–4000 ng/rat) dose-dependently reduced ethanol-induced gastric ulcers by 39–77%. Subcutaneous ghrelin administration (80 µg/kg) reduced ulcer depth only. L-NAME and capsaicin, but not vagotomy, prevented the gastroprotective effect of central ghrelin (4000 ng/rat).k4!1g, 百拇医药
This is the first evidence that ghrelin exerts a potent central gastroprotective activity against ethanol-induced lesions. The gastroprotective effect of ghrelin is mediated by endogenous NO release and requires the integrity of sensory nerve fibers.k4!1g, 百拇医药
Introductionk4!1g, 百拇医药
GHRELIN, the natural endogenous ligand for the GH secretagogue receptor (GHS-R), was originally isolated from the rat stomach (1) and was recently identified in endocrine cells of the gastrointestinal tract (2). Ghrelin immunoreactivity has also been detected in the hypothalamic arcuate nucleus (1). The finding that GHS-R are present in several brain areas and in peripheral tissues (3) indicates a regulatory role for the peptide in many biological activities.
Previous studies have shown that ghrelin stimulates GH release and food intake in rodents after either systemic or central administration (4, 5, 6, 7, 8). More interestingly, the finding that stomach ghrelin expression as well as plasma ghrelin levels increase during acute nutrient restriction (i.e. fasting and protein deprivation) bolsters a role for endogenous ghrelin in the regulation of food intake (9). As previously reported for many brain-gut peptides that influence feeding behavior, ghrelin can also trigger gastric secretory (10) and motor responses (11). An intriguing finding is that although ghrelin has been reported to stimulate gastric acid secretion in anesthetized rats (10, 11), we have recently demonstrated that the central administration of ghrelin effectively inhibits gastric acid secretion in conscious rats (12). The observation that in conscious rats the maximal acid inhibition by ghrelin is achieved at a dose much lower (1 pmol/rat) than that eliciting a stimulatory response (1 µmol/rat) strongly favors a physiological relevance for the inhibitory role of the peptide in the central regulation of gastric secretion. It is not known, however, whether the modulatory activity of ghrelin on gastric acid secretion might also be involved in the process of gastric mucosal injury and healing. As drugs releasing the potent vasodilator nitric oxide (NO) protect against mucosal injury (13), and the NO system is modulated by synthetic GHS-R agonists (14, 15), an interactive role of ghrelin and NO in controlling gastric mucosal blood flow may well be hypothesized. Previous studies have pointed to gastric microcirculatory disturbances as the main pathogenetic mechanism associated with ethanol-induced gastric lesions (16); therefore, our experiments were performed on this type of ulcer.
The aims of this study were 1) to examine the possible effects of centrally or peripherally administered ghrelin on acute hemorrhagic gastric mucosal injury, 2) to assess the possible interaction between ghrelin and NO system, and 3) to define the neural pathway(s) conveying the central effects of ghrelin to the stomach.(9z3a5, http://www.100md.com
Materials and Methods(9z3a5, http://www.100md.com
Animals(9z3a5, http://www.100md.com
Male Sprague Dawley rats, weighing 200–250 g (Charles River Laboratories, Inc., Calco, Italy), were housed in single cages that had wire-net bottoms to avoid coprophagy. Before the experiments all rats were starved for 24 h, but had free access to tap water until the beginning of treatment. Animals receiving intracerebroventicular (icv) treatment were implanted with a polyethylene cannula (PE10) in the left lateral ventricle, 5 d before the experiment as previously described (17). At the end of the experiment, dye (0.5% Evans blue) was injected through the cannula to confirm its position in the ventricle.
All procedures were performed out in accordance with the Italian Guidelines for the Use of Animals in Medical Research.46itn, 百拇医药
Drugs46itn, 百拇医药
Ghrelin was synthesized by conventional solid phase synthesis and was purified to at least 98% purity by HPLC (Neosystem, Strasbourg, France). The peptide was dissolved in saline immediately before the experiment and was injected in a volume of 5 µl/rat, icv, or 1 ml/kg, sc.46itn, 百拇医药
Absolute ethanol (EtOH; BDH, Poole, UK) was diluted 1:1 (vol/vol) in distilled water. Fifty percent EtOH was given in a volume of 1 ml/rat by an oral gavage. N{omega} -Nitro-L-arginine methyl ester (L-NAME; Sigma, St. Louis, MO) was dissolved in saline and administered sc at a dose of 70 mg/kg·1 ml. Capsaicin (Sigma) was dissolved in an appropriate vehicle (8% ethanol and 6% Tween 80 in physiological saline) and administered sc at the dosing volume of 2 ml.46itn, 百拇医药
Experimental procedures
Gastric ulcers and ghrelin.]mg3%, 百拇医药
Gastric mucosal damage was induced by oral administration of 1 ml 50% EtOH; ghrelin or saline was administered icv (4, 40, and 4000 ng/rat corresponding to 1, 10, and 1000 pmol/rat) or sc (40 and 80 µg/kg corresponding to 10 and 20 nmol/kg) 30 min before EtOH. The doses of ghrelin used were chosen on the basis of previous studies of gastric acid secretion (12).]mg3%, 百拇医药
NO and ghrelin.]mg3%, 百拇医药
The inhibitor of NO synthetase activity (L-NAME) was administered sc at a dose of 70 mg/kg 15 min before ghrelin (4000 ng/rat, icv) and 45 min before EtOH challenge accordingly to previous studies (18).]mg3%, 百拇医药
Vagotomy (vgx) and ghrelin.]mg3%, 百拇医药
Under ether anesthesia the cervical section of the vagus nerve was exposed, and bilateral cervical truncal vagotomy was performed. The vagus was similarly exposed in sham-operated control rats, but the vagal trunks were not sectioned. After closure of the incision, rats were allowed 3 h to recover from the operation before they were subjected to saline or ghrelin administration (4000 ng/rat, icv), followed 30 min later by EtOH administration.
Capsaicin-sensitive fibers and ghrelin..@t@ds, 百拇医药
Under ether anesthesia, rats were treated sc with increasing doses of 10, 20, 30, and 50 mg/kg capsaicin on 4 consecutive d according to the method reported by Izbéki et al. (19). Control rats received an equal volume of vehicle for capsaicin..@t@ds, 百拇医药
The effectiveness of the capsaicin denervation was assessed 7 d after the last capsaicin administration by instilling a drop of 1% NaOH solution into the left eye of each rat. Only rats with reduced wiping movements were used. The animals were challenged with EtOH and ghrelin (4000 ng/rat, icv) as described above..@t@ds, 百拇医药
Assessment of gastric mucosal lesions.@t@ds, 百拇医药
Macroscopic assessment..@t@ds, 百拇医药
Rats were euthanized with CO2 inhalation 1 h after EtOH exposure, and the stomachs were removed, opened along the lesser curvature, rinsed with saline, and examined for the severity and number of mucosal gastric lesions. The lesions were blindly examined according to a modified scoring system developed by Martin et al. (20) by two trained observers (V.S and C.N.). The average scores for each group were calculated and expressed as the ulcer index.
Microscopic assessment.;*cul2, 百拇医药
Histological evaluation was performed on the glandular stomach of two rats randomly selected from representative experiments. In brief, stomachs were fixed by immersion in 4% buffered formalin, correctly oriented, embedded in paraffin, and cut. Serial paraffin sections (2–4 µm) were hydrated and stained with hematoxylin and eosin for mucosal damage assessment or with sera specific for ghrelin, gastrin, and somatostatin as previously described (21) for endocrine cell assessment. The mucosal injury evaluation was performed under light microscopy on correctly oriented mucosa by two of the authors in a blinded fashion (G.R. and N.C.) and was quantified according to a two-step scoring system (length score and depth score). First, the extension of the mucosal lesion was measured and expressed as a percentage of the entire mucosa investigated. Accordingly a partial score from 0–3 was assigned (0 = no lesion; 1 = lesion involving 1–10% of the mucosa; 2 = lesion involving 11–20% of the mucosa; 3 = lesion involving >20% of the mucosa). Second, the deepest mucosal lesion was identified per stomach sample and scored from 0–3 (0 = no change; 0.5 = superficial erosion; 1 = ulcer involving one internal third of the mucosa; 2 = ulcer involving the two internal thirds of the mucosa; 3 = ulcer involving almost the entire mucosal thickness). The total microscopic score resulted by the sum of the two partial scores and ranged from 0–6.
Statistical analysis^, 百拇医药
Statistical analysis was performed with a statistic package (PRISM, GraphPad Software, Inc., San Diego, CA). All data are represented as the mean ± SEM. Differences between groups were assessed by means of nonparametric statistical analysis: Kruskal-Wallis test, followed by multicomparison Dunn’s test. The percent decrease in gastric lesions was calculated by comparison with control group values. The ED50 was calculated by standard regression analysis of the dose-response data. Linear regression analysis was also used to assess the correlation between macro- and microevaluations. P < 0.05 was considered significant.^, 百拇医药
Results^, 百拇医药
Ghrelin is a potent inhibitor of EtOH-induced gastric injury^, 百拇医药
The effects of icv injections of ghrelin on gastric ulcers induced by EtOH and assessed by macroscopical evaluation are shown in Fig. 1. Ghrelin exhibited a dose-dependent protective effect against EtOH-induced gastric ulcers, with a significant inhibition compared with the saline group at a dose of 40 ng/rat (-39%) and maximal inhibition at a dose of 4000 ng/rat (-77%). The ED50 value calculated from the log dose-response curve was 115 ng/rat. The sc administration of ghrelin (40 and 80 µg/kg) did not induce a significant protective effect against EtOH-induced gastric lesions. The ulcer macroscopic scores were 4.42 ± 0.17 in the controls and 4.1 ± 0.35 and 3.3 ± 0.65 in the animals treated with 40 and 80 µg/kg ghrelin, respectively. Nonetheless, some protective effect of ghrelin sc (80 µg/kg) was detected at histology. Histological results are reported in Table 1 and Fig. 2. A mean of 67 ± 22 mm of mucosa was evaluated per rat. EtOH administration induced severe lesions (Table 1) characterized by coagulative necrosis of the glands with diffuse hemorrhage of the mucosa (Fig. 2, A and B). Rats treated with 4000 ng ghrelin, icv, showed normal histology (Fig. 2C) or very superficial lesions only (Fig. 2D). Rats treated with 80 µg/kg ghrelin, sc, displayed a length score similar to that of EtOH-saline treated rats, but a reduced depth score (Table 1 and Fig. 2E). Statistical analysis performed on macro- and microscopic total scores revealed an excellent correlation (Fig. 2F), thus confirming the effectiveness of the macroscopic evaluation. Overall, the above data indicate a potent protective effect against EtOH-induced gastric lesions by central ghrelin and a partial peripheral protective effect.
fig.ommitteed#., 百拇医药
Figure 1. Effects of different doses of ghrelin injected icv (30 min before) on gastric ulcers induced by 1 ml 50% (vol/vol) EtOH, orally, in conscious rats. Gastric lesions were monitored 60 min after EtOH treatment. Each bar represents the mean ± SEM of 8–12 animals. *, P < 0.05; **, P < 0.01 (vs. saline group). Inset, Linear regression analysis performed on data expressed as a percentage of the ulcer index inhibition of controls.#., 百拇医药
fig.ommitteed#., 百拇医药
Table 1. Microscopical assessment of EtOH-induced lesions in gastric sections from rats treated with saline or ghrelin#., 百拇医药
fig.ommitteed#., 百拇医药
Figure 2. Histological assessment of the oxyntic mucosa after EtOH acute challenge in rats treated with saline (A and B) or ghrelin (C–E). A–E, Arrowheads indicate the lesion borders; vertical bars are given as a reference for mucosal thickness; asterisks indicate the gastric lumen. After acute EtOH challenge and icv saline, deep gastric erosions involve almost the entire mucosal layer (A; microscopic grade 3) or about two thirds of the thickness (B; microscopic grade 2). Note the effacement of the mucosal architecture with cell necrosis and diffuse hemorrhage (center of the micrographs, above arrowheads; A and B). After EtOH acute challenge and 4 µg ghrelin, icv, the gastric mucosa is normal (C) or displays only slight, superficial damage (D; microscopic grade 0.5). The insets show the normal morphology of foveolar cells (C) and coagulative cell necrosis (D). Peripheral ghrelin (80 µg/kg, sc) administration partially prevented the effects of acute EtOH challenge, as shown by the superficial ulcer involving only the inner third of the mucosa (E; microscopic grade 1); note the coagulative necrosis with fibrin deposition at the surface (above arrowheads). Hematoxylin and eosin stain. Magnification: A–E, x180; insets, x300. F, Correlation curve of microscopic (MiS) vs. macroscopic (MaS) scores.
Protective effects of centrally administered ghrelin are mediated by NOlj:u\y$, http://www.100md.com
Pretreatment of rats with the inhibitor of NO synthase, L-NAME (70 mg/kg, sc), slightly increased the severity of gastric lesions induced by EtOH and completely abolished the protective effect of ghrelin (4000 ng/rat, icv; Fig. 3). Histological evaluation confirmed the effectiveness of L-NAME in removing the gastroprotective effect of ghrelin (Table 1). The above data indicate that the gastroprotective effect of centrally administered ghrelin is mediated by NO pathways.lj:u\y$, http://www.100md.com
fig.ommitteedlj:u\y$, http://www.100md.com
Figure 3. Effect of pretreatment (15 min before) with L-NAME (70 mg/kg, sc) on the gastroprotective effect of ghrelin (4000 ng/rat, icv) given 30 min before inducing gastric ulcers by 1 ml 50% (vol/vol) EtOH, orally, in conscious rats. Gastric lesions were monitored 60 min after EtOH treatment. Each bar represents the mean ± SEM of 8–12 animals. **, P < 0.01 vs. saline; °°, P < 0.01 vs. saline and ghrelin.lj:u\y$, http://www.100md.com
Centrally administered ghrelin acts via sensory neurons and not via the vagus)62t.ne, http://www.100md.com
Figure 4 reports the results obtained in vgx rats. As expected, acute bilateral cervical vgx worsened, even if not significantly, the extent of EtOH-induced gastric damage. Vagotomy failed to affect the gastroprotective activity of ghrelin (4000 ng/rat, icv), as the percent ghrelin inhibition of EtOH-induced gastric lesions was similar in vgx (-60%) and sham-operated (-69%) rats. Similarly, at histology only mild lesions were observed in vgx, ghrelin-treated rats compared with vgx, saline-treated or sham-operated, saline-treated rats (Table 1). These data indicate that centrally administered ghrelin does not exert its protective role on gastric mucosa via the vagus nerve.)62t.ne, http://www.100md.com
fig.ommitteed)62t.ne, http://www.100md.com
Figure 4. Effect of ghrelin (4000 ng/rat, icv) given 30 min before inducing gastric ulcers by administration of 1 ml 50% (vol/vol) EtOH, orally, in conscious sham-operated and vgx rats. Acute bilateral cervical vgx or sham operation was performed 3 h before icv injection of ghrelin. Gastric lesions were monitored 60 min after EtOH treatment. Each bar represents the mean ± SEM of six to eight animals. *, P < 0.05 vs. saline-treated sham operated or vgx rats.
To explore the possible role of capsaicin-sensitive sensory fibers on the ulcer-protective effect of ghrelin, rats were pretreated with capsaicin. Capsaicin treatment significantly worsened EtOH-induced gastric lesions (+22%) compared with those in vehicle-pretreated rats and completely prevented the gastroprotective effects of centrally administered ghrelin (4000 ng/rat, icv; Fig. 5). After histological analysis, capsaicin- and ghrelin-treated rats exhibited lesions comparable to those in capsaicin- and saline-treated or vehicle- and saline-treated rats (Table 1). These data suggest that capsaicin-sensitive sensory nerve fibers are involved in the central gastroprotective effect of ghrelin.]o9l|, 百拇医药
fig.ommitteed]o9l|, 百拇医药
Figure 5. Effect of ghrelin (4000 ng/rat, icv) given 30 min before inducing gastric ulcers by administration of 1 ml 50% (vol/vol) EtOH, orally, in conscious vehicle-treated rats with intact sensory nerves and in those with capsaicin-induced sensory denervation. Gastric lesions were monitored 60 min after EtOH treatment. Each bar represents the mean ± SEM of 6–10 animals. *, P < 0.05 vs. vehicle; °, P < 0.05 vs. vehicle and ghrelin.
Gastric endocrine cells are not modified after ghrelin administration'jp, 百拇医药
To assess gastric endocrine cell change, we investigated by immunohistochemistry ghrelin, somatostatin, and gastrin cells of the gastric mucosa. The qualitative assessment of size, shape, and number of ghrelin, somatostatin, and gastrin cells showed no change compared with untreated rats (21) in any of the above-described experiments, except when the gastric mucosa was deeply ulcerated (not shown). This observation supports the view that the effect of ghrelin on EtOH-induced gastric lesions is apparently not mediated by local endocrine cell changes.'jp, 百拇医药
Discussion'jp, 百拇医药
The present study demonstrates for the first time that centrally administered ghrelin exerts a dose-dependent gastroprotective effect on acute, EtOH-induced gastric lesions in the rat. Moreover, our data indicate that the protective effect of ghrelin is mediated by NO and that capsaicin sensory nerves, and not the vagus, convey the central impulses of ghrelin, resulting in gastric mucosal protection.
A recent functional mapping study has shown that acute central administration of ghrelin in rats induces the immediate-early gene c-fos, a marker of neuronal activation, in several hypothalamic nuclei and in the brainstem (22). Moreover, the evidence that ghrelin recognizes the receptors for GHS synthetic peptides (23) and that GHS-R mRNA is located in the same brain regions where c-fos expression has been observed (3) strongly supports the hypothesis that ghrelin can directly activate signaling from the brain down to the stomach. Interestingly, the icv dose of ghrelin (0.1 µg) reported to activate c-fos in the rat brain (22) is comparable to the ED50 value (0.115 µg/rat) calculated by us for the gastroprotective effect of the peptide. Moreover, the evidence that the doses of ghrelin used are much lower than those reported to saturate the transport system for ghrelin from the brain to blood (24) suggests a physiological relevance for the central gastroprotective effect of ghrelin.
The gastroprotective effect of centrally administered ghrelin appears to be mediated by the NO pathway. Indeed, inhibition of NO synthesis by L-NAME completely abolished the gastroprotective effect of ghrelin. Consistent with these data, an interaction between synthetic GHS and the NO system has been previously reported (14, 15). In fact, NO-releasing drugs protect against EtOH-induced gastric lesions, and conversely, inhibition of NO synthesis increases the susceptibility of the stomach to ethanol injury (13). Furthermore, considering that NO plays a role in the ulcer repair process (25), it will be interesting to examine the potential activity of ghrelin in the course of healing of chronic gastric ulcers.48c2v|, 百拇医药
It is unlikely that the gastric-protective effect of ghrelin involves an interaction with ghrelin-binding sites in the brain stem (nucleus of tractus solitarius and dorsomotor nucleus of the vagus), because these nuclei act as relay stations that receive and send information to the stomach through vagal pathways (26). Our data, in fact, show that ghrelin retains its gastric-protective effect after bilateral vagotomy, thus indicating that the brain stem vagal complex is not involved in the gastroprotective effect of centrally administered ghrelin.
Previous reports demonstrated that icv or systemic ghrelin induced gastric acid secretion, and this effect was abolished by vagotomy (11). In contrast, we found that ghrelin, as reported for other peptides [e.g. neuropeptide Y (27)], displays central inhibitory activity on gastric acid secretion (12). This dual effect is probably due to the different neuronal pathways activated by ghrelin. In fact, the lateral and paraventricular hypothalamic nuclei, containing a high density of ghrelin-binding sites (3), influence the gastrointestinal system through both parasympathetic and sympathetic pathways (28, 29). The present data indicate that ghrelin elicits its maximal protective effect on EtOH-induced lesions at a dosage 1000-fold higher than the dosage previously shown to exert a maximal inhibition on gastric acid secretion. Therefore, it appears that the gastroprotective effect of ghrelin is not strictly related to its inhibitory activity on gastric acid secretion. Indeed, the mucosal damage induced by EtOH is independent of gastric acid secretion (13, 16). Accordingly, various peptides, when centrally injected, may protect against EtOH-induced gastric lesions by activating neural pathways, ultimately releasing endogenous mucosal protective substances in the stomach (30, 31, 32). The rodent gastric wall receives a dense supply by spinal afferent nerve fibers expressing sensory neuropeptides [e.g. calcitonin gene-related peptide (CGRP) and substance P] and neuronal messengers (NO), which are all involved in mucosal defense (26). The present findings show that capsaicin ablation of primary sensory neurons results in exacerbation of EtOH-induced gastric lesions. In such conditions, ghrelin failed to enhance the resistance of the mucosa to EtOH-induced gastric injury. This implies that the central gastroprotective activity of ghrelin relies on the integrity of spinal sensory nerve fibers. It is known that capsaicin treatment induces complete depletion of CGRP-containing fibers (33), whereas other peptidergic fibers are only reduced (substance P and pituitary adenylate cyclase-activating polypeptide) (34, 35) or unchanged (vasoactive intestinal peptide) (36). Therefore, it could be speculated that the central gastroprotective effect of ghrelin is mediated by CGRP fibers. This view is consistent with the evidence that NO acts as a secondary messenger of CGRP function in mucosal homeostasis (37) and by the present data showing that blockade of NO synthase by L-NAME compromises the gastroprotective activity of ghrelin.
Finally, we have demonstrated that sc administration of 80 µg/kg ghrelin still retains some minor gastroprotective effect, as demonstrated by histology. The peripheral gastroprotective effect of the peptide could involve the beneficial effects on hemodynamic parameters recently documented for ghrelin in both humans (38) and rats (39). However, a central site of action for the gastroprotective effect of sc ghrelin cannot be excluded, because activation of hypothalamic arcuate nucleus was detected when the peptide was administered iv in the rat (40).l, http://www.100md.com
In conclusion, this is the first evidence that ghrelin exerts a potent gastroprotective activity against EtOH-induced lesions, mainly after central administration. The gastroprotective effect of ghrelin is mediated by endogenous NO release and requires the integrity of sensory nerve fibers. In gastroenterological practice, acute hemorrhagic and erosive gastropathy may represent a potentially life-threatening condition (41), often requiring urgent surgery. Ghrelin deserves further attention because it could represent a new interesting pharmacological tool for the treatment of acute erosive gastropathy.
Received July 24, 2002.&&, http://www.100md.com
Accepted for publication October 1, 2002.&&, http://www.100md.com
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Yardly JH, Hendrix TR 1999 Gastritis, gastropathy, duodenitis and associated ulcerative lesions. In: Yamada T, ed. Textbook of Gastroenterology. 3rd ed. Philadelphia: Lippincott, Williams & Wilkins; vol 11:1463–1499(V. Sibilia G. Rindi F. Pagani D. Rapetti V. Locatelli A. Torsello N. Campanini R. Deghenghi and C. N)
Ghrelin, the endogenous ligand for GH secretagogue receptors, has been reported to influence acid gastric secretion and motility, but its potential gastroprotective effect is unknown. The aims of this study were 1) to examine the effects of central and peripheral administration of ghrelin on ethanol-induced gastric ulcers in conscious rats, and 2) to investigate the possible roles of nitric oxide (NO), vagal nerve, and sensory fibers in the gastric effects of ghrelin.q/#r*, 百拇医药
Ghrelin was administered either intracerebroventricularly or sc 30 min before ethanol, and mucosal lesions were examined macroscopically. Additionally, rats were either treated with the inhibitor of NO synthesis N{omega} -nitro-L-arginine methyl ester (L-NAME) or underwent bilateral cervical vagotomy or capsaicin-induced sensory denervation. Conventional histology and immunohistochemistry for ghrelin, gastrin, and somatostatin were performed on gastric specimens from representative rats.
Central ghrelin (4–4000 ng/rat) dose-dependently reduced ethanol-induced gastric ulcers by 39–77%. Subcutaneous ghrelin administration (80 µg/kg) reduced ulcer depth only. L-NAME and capsaicin, but not vagotomy, prevented the gastroprotective effect of central ghrelin (4000 ng/rat).k4!1g, 百拇医药
This is the first evidence that ghrelin exerts a potent central gastroprotective activity against ethanol-induced lesions. The gastroprotective effect of ghrelin is mediated by endogenous NO release and requires the integrity of sensory nerve fibers.k4!1g, 百拇医药
Introductionk4!1g, 百拇医药
GHRELIN, the natural endogenous ligand for the GH secretagogue receptor (GHS-R), was originally isolated from the rat stomach (1) and was recently identified in endocrine cells of the gastrointestinal tract (2). Ghrelin immunoreactivity has also been detected in the hypothalamic arcuate nucleus (1). The finding that GHS-R are present in several brain areas and in peripheral tissues (3) indicates a regulatory role for the peptide in many biological activities.
Previous studies have shown that ghrelin stimulates GH release and food intake in rodents after either systemic or central administration (4, 5, 6, 7, 8). More interestingly, the finding that stomach ghrelin expression as well as plasma ghrelin levels increase during acute nutrient restriction (i.e. fasting and protein deprivation) bolsters a role for endogenous ghrelin in the regulation of food intake (9). As previously reported for many brain-gut peptides that influence feeding behavior, ghrelin can also trigger gastric secretory (10) and motor responses (11). An intriguing finding is that although ghrelin has been reported to stimulate gastric acid secretion in anesthetized rats (10, 11), we have recently demonstrated that the central administration of ghrelin effectively inhibits gastric acid secretion in conscious rats (12). The observation that in conscious rats the maximal acid inhibition by ghrelin is achieved at a dose much lower (1 pmol/rat) than that eliciting a stimulatory response (1 µmol/rat) strongly favors a physiological relevance for the inhibitory role of the peptide in the central regulation of gastric secretion. It is not known, however, whether the modulatory activity of ghrelin on gastric acid secretion might also be involved in the process of gastric mucosal injury and healing. As drugs releasing the potent vasodilator nitric oxide (NO) protect against mucosal injury (13), and the NO system is modulated by synthetic GHS-R agonists (14, 15), an interactive role of ghrelin and NO in controlling gastric mucosal blood flow may well be hypothesized. Previous studies have pointed to gastric microcirculatory disturbances as the main pathogenetic mechanism associated with ethanol-induced gastric lesions (16); therefore, our experiments were performed on this type of ulcer.
The aims of this study were 1) to examine the possible effects of centrally or peripherally administered ghrelin on acute hemorrhagic gastric mucosal injury, 2) to assess the possible interaction between ghrelin and NO system, and 3) to define the neural pathway(s) conveying the central effects of ghrelin to the stomach.(9z3a5, http://www.100md.com
Materials and Methods(9z3a5, http://www.100md.com
Animals(9z3a5, http://www.100md.com
Male Sprague Dawley rats, weighing 200–250 g (Charles River Laboratories, Inc., Calco, Italy), were housed in single cages that had wire-net bottoms to avoid coprophagy. Before the experiments all rats were starved for 24 h, but had free access to tap water until the beginning of treatment. Animals receiving intracerebroventicular (icv) treatment were implanted with a polyethylene cannula (PE10) in the left lateral ventricle, 5 d before the experiment as previously described (17). At the end of the experiment, dye (0.5% Evans blue) was injected through the cannula to confirm its position in the ventricle.
All procedures were performed out in accordance with the Italian Guidelines for the Use of Animals in Medical Research.46itn, 百拇医药
Drugs46itn, 百拇医药
Ghrelin was synthesized by conventional solid phase synthesis and was purified to at least 98% purity by HPLC (Neosystem, Strasbourg, France). The peptide was dissolved in saline immediately before the experiment and was injected in a volume of 5 µl/rat, icv, or 1 ml/kg, sc.46itn, 百拇医药
Absolute ethanol (EtOH; BDH, Poole, UK) was diluted 1:1 (vol/vol) in distilled water. Fifty percent EtOH was given in a volume of 1 ml/rat by an oral gavage. N{omega} -Nitro-L-arginine methyl ester (L-NAME; Sigma, St. Louis, MO) was dissolved in saline and administered sc at a dose of 70 mg/kg·1 ml. Capsaicin (Sigma) was dissolved in an appropriate vehicle (8% ethanol and 6% Tween 80 in physiological saline) and administered sc at the dosing volume of 2 ml.46itn, 百拇医药
Experimental procedures
Gastric ulcers and ghrelin.]mg3%, 百拇医药
Gastric mucosal damage was induced by oral administration of 1 ml 50% EtOH; ghrelin or saline was administered icv (4, 40, and 4000 ng/rat corresponding to 1, 10, and 1000 pmol/rat) or sc (40 and 80 µg/kg corresponding to 10 and 20 nmol/kg) 30 min before EtOH. The doses of ghrelin used were chosen on the basis of previous studies of gastric acid secretion (12).]mg3%, 百拇医药
NO and ghrelin.]mg3%, 百拇医药
The inhibitor of NO synthetase activity (L-NAME) was administered sc at a dose of 70 mg/kg 15 min before ghrelin (4000 ng/rat, icv) and 45 min before EtOH challenge accordingly to previous studies (18).]mg3%, 百拇医药
Vagotomy (vgx) and ghrelin.]mg3%, 百拇医药
Under ether anesthesia the cervical section of the vagus nerve was exposed, and bilateral cervical truncal vagotomy was performed. The vagus was similarly exposed in sham-operated control rats, but the vagal trunks were not sectioned. After closure of the incision, rats were allowed 3 h to recover from the operation before they were subjected to saline or ghrelin administration (4000 ng/rat, icv), followed 30 min later by EtOH administration.
Capsaicin-sensitive fibers and ghrelin..@t@ds, 百拇医药
Under ether anesthesia, rats were treated sc with increasing doses of 10, 20, 30, and 50 mg/kg capsaicin on 4 consecutive d according to the method reported by Izbéki et al. (19). Control rats received an equal volume of vehicle for capsaicin..@t@ds, 百拇医药
The effectiveness of the capsaicin denervation was assessed 7 d after the last capsaicin administration by instilling a drop of 1% NaOH solution into the left eye of each rat. Only rats with reduced wiping movements were used. The animals were challenged with EtOH and ghrelin (4000 ng/rat, icv) as described above..@t@ds, 百拇医药
Assessment of gastric mucosal lesions.@t@ds, 百拇医药
Macroscopic assessment..@t@ds, 百拇医药
Rats were euthanized with CO2 inhalation 1 h after EtOH exposure, and the stomachs were removed, opened along the lesser curvature, rinsed with saline, and examined for the severity and number of mucosal gastric lesions. The lesions were blindly examined according to a modified scoring system developed by Martin et al. (20) by two trained observers (V.S and C.N.). The average scores for each group were calculated and expressed as the ulcer index.
Microscopic assessment.;*cul2, 百拇医药
Histological evaluation was performed on the glandular stomach of two rats randomly selected from representative experiments. In brief, stomachs were fixed by immersion in 4% buffered formalin, correctly oriented, embedded in paraffin, and cut. Serial paraffin sections (2–4 µm) were hydrated and stained with hematoxylin and eosin for mucosal damage assessment or with sera specific for ghrelin, gastrin, and somatostatin as previously described (21) for endocrine cell assessment. The mucosal injury evaluation was performed under light microscopy on correctly oriented mucosa by two of the authors in a blinded fashion (G.R. and N.C.) and was quantified according to a two-step scoring system (length score and depth score). First, the extension of the mucosal lesion was measured and expressed as a percentage of the entire mucosa investigated. Accordingly a partial score from 0–3 was assigned (0 = no lesion; 1 = lesion involving 1–10% of the mucosa; 2 = lesion involving 11–20% of the mucosa; 3 = lesion involving >20% of the mucosa). Second, the deepest mucosal lesion was identified per stomach sample and scored from 0–3 (0 = no change; 0.5 = superficial erosion; 1 = ulcer involving one internal third of the mucosa; 2 = ulcer involving the two internal thirds of the mucosa; 3 = ulcer involving almost the entire mucosal thickness). The total microscopic score resulted by the sum of the two partial scores and ranged from 0–6.
Statistical analysis^, 百拇医药
Statistical analysis was performed with a statistic package (PRISM, GraphPad Software, Inc., San Diego, CA). All data are represented as the mean ± SEM. Differences between groups were assessed by means of nonparametric statistical analysis: Kruskal-Wallis test, followed by multicomparison Dunn’s test. The percent decrease in gastric lesions was calculated by comparison with control group values. The ED50 was calculated by standard regression analysis of the dose-response data. Linear regression analysis was also used to assess the correlation between macro- and microevaluations. P < 0.05 was considered significant.^, 百拇医药
Results^, 百拇医药
Ghrelin is a potent inhibitor of EtOH-induced gastric injury^, 百拇医药
The effects of icv injections of ghrelin on gastric ulcers induced by EtOH and assessed by macroscopical evaluation are shown in Fig. 1. Ghrelin exhibited a dose-dependent protective effect against EtOH-induced gastric ulcers, with a significant inhibition compared with the saline group at a dose of 40 ng/rat (-39%) and maximal inhibition at a dose of 4000 ng/rat (-77%). The ED50 value calculated from the log dose-response curve was 115 ng/rat. The sc administration of ghrelin (40 and 80 µg/kg) did not induce a significant protective effect against EtOH-induced gastric lesions. The ulcer macroscopic scores were 4.42 ± 0.17 in the controls and 4.1 ± 0.35 and 3.3 ± 0.65 in the animals treated with 40 and 80 µg/kg ghrelin, respectively. Nonetheless, some protective effect of ghrelin sc (80 µg/kg) was detected at histology. Histological results are reported in Table 1 and Fig. 2. A mean of 67 ± 22 mm of mucosa was evaluated per rat. EtOH administration induced severe lesions (Table 1) characterized by coagulative necrosis of the glands with diffuse hemorrhage of the mucosa (Fig. 2, A and B). Rats treated with 4000 ng ghrelin, icv, showed normal histology (Fig. 2C) or very superficial lesions only (Fig. 2D). Rats treated with 80 µg/kg ghrelin, sc, displayed a length score similar to that of EtOH-saline treated rats, but a reduced depth score (Table 1 and Fig. 2E). Statistical analysis performed on macro- and microscopic total scores revealed an excellent correlation (Fig. 2F), thus confirming the effectiveness of the macroscopic evaluation. Overall, the above data indicate a potent protective effect against EtOH-induced gastric lesions by central ghrelin and a partial peripheral protective effect.
fig.ommitteed#., 百拇医药
Figure 1. Effects of different doses of ghrelin injected icv (30 min before) on gastric ulcers induced by 1 ml 50% (vol/vol) EtOH, orally, in conscious rats. Gastric lesions were monitored 60 min after EtOH treatment. Each bar represents the mean ± SEM of 8–12 animals. *, P < 0.05; **, P < 0.01 (vs. saline group). Inset, Linear regression analysis performed on data expressed as a percentage of the ulcer index inhibition of controls.#., 百拇医药
fig.ommitteed#., 百拇医药
Table 1. Microscopical assessment of EtOH-induced lesions in gastric sections from rats treated with saline or ghrelin#., 百拇医药
fig.ommitteed#., 百拇医药
Figure 2. Histological assessment of the oxyntic mucosa after EtOH acute challenge in rats treated with saline (A and B) or ghrelin (C–E). A–E, Arrowheads indicate the lesion borders; vertical bars are given as a reference for mucosal thickness; asterisks indicate the gastric lumen. After acute EtOH challenge and icv saline, deep gastric erosions involve almost the entire mucosal layer (A; microscopic grade 3) or about two thirds of the thickness (B; microscopic grade 2). Note the effacement of the mucosal architecture with cell necrosis and diffuse hemorrhage (center of the micrographs, above arrowheads; A and B). After EtOH acute challenge and 4 µg ghrelin, icv, the gastric mucosa is normal (C) or displays only slight, superficial damage (D; microscopic grade 0.5). The insets show the normal morphology of foveolar cells (C) and coagulative cell necrosis (D). Peripheral ghrelin (80 µg/kg, sc) administration partially prevented the effects of acute EtOH challenge, as shown by the superficial ulcer involving only the inner third of the mucosa (E; microscopic grade 1); note the coagulative necrosis with fibrin deposition at the surface (above arrowheads). Hematoxylin and eosin stain. Magnification: A–E, x180; insets, x300. F, Correlation curve of microscopic (MiS) vs. macroscopic (MaS) scores.
Protective effects of centrally administered ghrelin are mediated by NOlj:u\y$, http://www.100md.com
Pretreatment of rats with the inhibitor of NO synthase, L-NAME (70 mg/kg, sc), slightly increased the severity of gastric lesions induced by EtOH and completely abolished the protective effect of ghrelin (4000 ng/rat, icv; Fig. 3). Histological evaluation confirmed the effectiveness of L-NAME in removing the gastroprotective effect of ghrelin (Table 1). The above data indicate that the gastroprotective effect of centrally administered ghrelin is mediated by NO pathways.lj:u\y$, http://www.100md.com
fig.ommitteedlj:u\y$, http://www.100md.com
Figure 3. Effect of pretreatment (15 min before) with L-NAME (70 mg/kg, sc) on the gastroprotective effect of ghrelin (4000 ng/rat, icv) given 30 min before inducing gastric ulcers by 1 ml 50% (vol/vol) EtOH, orally, in conscious rats. Gastric lesions were monitored 60 min after EtOH treatment. Each bar represents the mean ± SEM of 8–12 animals. **, P < 0.01 vs. saline; °°, P < 0.01 vs. saline and ghrelin.lj:u\y$, http://www.100md.com
Centrally administered ghrelin acts via sensory neurons and not via the vagus)62t.ne, http://www.100md.com
Figure 4 reports the results obtained in vgx rats. As expected, acute bilateral cervical vgx worsened, even if not significantly, the extent of EtOH-induced gastric damage. Vagotomy failed to affect the gastroprotective activity of ghrelin (4000 ng/rat, icv), as the percent ghrelin inhibition of EtOH-induced gastric lesions was similar in vgx (-60%) and sham-operated (-69%) rats. Similarly, at histology only mild lesions were observed in vgx, ghrelin-treated rats compared with vgx, saline-treated or sham-operated, saline-treated rats (Table 1). These data indicate that centrally administered ghrelin does not exert its protective role on gastric mucosa via the vagus nerve.)62t.ne, http://www.100md.com
fig.ommitteed)62t.ne, http://www.100md.com
Figure 4. Effect of ghrelin (4000 ng/rat, icv) given 30 min before inducing gastric ulcers by administration of 1 ml 50% (vol/vol) EtOH, orally, in conscious sham-operated and vgx rats. Acute bilateral cervical vgx or sham operation was performed 3 h before icv injection of ghrelin. Gastric lesions were monitored 60 min after EtOH treatment. Each bar represents the mean ± SEM of six to eight animals. *, P < 0.05 vs. saline-treated sham operated or vgx rats.
To explore the possible role of capsaicin-sensitive sensory fibers on the ulcer-protective effect of ghrelin, rats were pretreated with capsaicin. Capsaicin treatment significantly worsened EtOH-induced gastric lesions (+22%) compared with those in vehicle-pretreated rats and completely prevented the gastroprotective effects of centrally administered ghrelin (4000 ng/rat, icv; Fig. 5). After histological analysis, capsaicin- and ghrelin-treated rats exhibited lesions comparable to those in capsaicin- and saline-treated or vehicle- and saline-treated rats (Table 1). These data suggest that capsaicin-sensitive sensory nerve fibers are involved in the central gastroprotective effect of ghrelin.]o9l|, 百拇医药
fig.ommitteed]o9l|, 百拇医药
Figure 5. Effect of ghrelin (4000 ng/rat, icv) given 30 min before inducing gastric ulcers by administration of 1 ml 50% (vol/vol) EtOH, orally, in conscious vehicle-treated rats with intact sensory nerves and in those with capsaicin-induced sensory denervation. Gastric lesions were monitored 60 min after EtOH treatment. Each bar represents the mean ± SEM of 6–10 animals. *, P < 0.05 vs. vehicle; °, P < 0.05 vs. vehicle and ghrelin.
Gastric endocrine cells are not modified after ghrelin administration'jp, 百拇医药
To assess gastric endocrine cell change, we investigated by immunohistochemistry ghrelin, somatostatin, and gastrin cells of the gastric mucosa. The qualitative assessment of size, shape, and number of ghrelin, somatostatin, and gastrin cells showed no change compared with untreated rats (21) in any of the above-described experiments, except when the gastric mucosa was deeply ulcerated (not shown). This observation supports the view that the effect of ghrelin on EtOH-induced gastric lesions is apparently not mediated by local endocrine cell changes.'jp, 百拇医药
Discussion'jp, 百拇医药
The present study demonstrates for the first time that centrally administered ghrelin exerts a dose-dependent gastroprotective effect on acute, EtOH-induced gastric lesions in the rat. Moreover, our data indicate that the protective effect of ghrelin is mediated by NO and that capsaicin sensory nerves, and not the vagus, convey the central impulses of ghrelin, resulting in gastric mucosal protection.
A recent functional mapping study has shown that acute central administration of ghrelin in rats induces the immediate-early gene c-fos, a marker of neuronal activation, in several hypothalamic nuclei and in the brainstem (22). Moreover, the evidence that ghrelin recognizes the receptors for GHS synthetic peptides (23) and that GHS-R mRNA is located in the same brain regions where c-fos expression has been observed (3) strongly supports the hypothesis that ghrelin can directly activate signaling from the brain down to the stomach. Interestingly, the icv dose of ghrelin (0.1 µg) reported to activate c-fos in the rat brain (22) is comparable to the ED50 value (0.115 µg/rat) calculated by us for the gastroprotective effect of the peptide. Moreover, the evidence that the doses of ghrelin used are much lower than those reported to saturate the transport system for ghrelin from the brain to blood (24) suggests a physiological relevance for the central gastroprotective effect of ghrelin.
The gastroprotective effect of centrally administered ghrelin appears to be mediated by the NO pathway. Indeed, inhibition of NO synthesis by L-NAME completely abolished the gastroprotective effect of ghrelin. Consistent with these data, an interaction between synthetic GHS and the NO system has been previously reported (14, 15). In fact, NO-releasing drugs protect against EtOH-induced gastric lesions, and conversely, inhibition of NO synthesis increases the susceptibility of the stomach to ethanol injury (13). Furthermore, considering that NO plays a role in the ulcer repair process (25), it will be interesting to examine the potential activity of ghrelin in the course of healing of chronic gastric ulcers.48c2v|, 百拇医药
It is unlikely that the gastric-protective effect of ghrelin involves an interaction with ghrelin-binding sites in the brain stem (nucleus of tractus solitarius and dorsomotor nucleus of the vagus), because these nuclei act as relay stations that receive and send information to the stomach through vagal pathways (26). Our data, in fact, show that ghrelin retains its gastric-protective effect after bilateral vagotomy, thus indicating that the brain stem vagal complex is not involved in the gastroprotective effect of centrally administered ghrelin.
Previous reports demonstrated that icv or systemic ghrelin induced gastric acid secretion, and this effect was abolished by vagotomy (11). In contrast, we found that ghrelin, as reported for other peptides [e.g. neuropeptide Y (27)], displays central inhibitory activity on gastric acid secretion (12). This dual effect is probably due to the different neuronal pathways activated by ghrelin. In fact, the lateral and paraventricular hypothalamic nuclei, containing a high density of ghrelin-binding sites (3), influence the gastrointestinal system through both parasympathetic and sympathetic pathways (28, 29). The present data indicate that ghrelin elicits its maximal protective effect on EtOH-induced lesions at a dosage 1000-fold higher than the dosage previously shown to exert a maximal inhibition on gastric acid secretion. Therefore, it appears that the gastroprotective effect of ghrelin is not strictly related to its inhibitory activity on gastric acid secretion. Indeed, the mucosal damage induced by EtOH is independent of gastric acid secretion (13, 16). Accordingly, various peptides, when centrally injected, may protect against EtOH-induced gastric lesions by activating neural pathways, ultimately releasing endogenous mucosal protective substances in the stomach (30, 31, 32). The rodent gastric wall receives a dense supply by spinal afferent nerve fibers expressing sensory neuropeptides [e.g. calcitonin gene-related peptide (CGRP) and substance P] and neuronal messengers (NO), which are all involved in mucosal defense (26). The present findings show that capsaicin ablation of primary sensory neurons results in exacerbation of EtOH-induced gastric lesions. In such conditions, ghrelin failed to enhance the resistance of the mucosa to EtOH-induced gastric injury. This implies that the central gastroprotective activity of ghrelin relies on the integrity of spinal sensory nerve fibers. It is known that capsaicin treatment induces complete depletion of CGRP-containing fibers (33), whereas other peptidergic fibers are only reduced (substance P and pituitary adenylate cyclase-activating polypeptide) (34, 35) or unchanged (vasoactive intestinal peptide) (36). Therefore, it could be speculated that the central gastroprotective effect of ghrelin is mediated by CGRP fibers. This view is consistent with the evidence that NO acts as a secondary messenger of CGRP function in mucosal homeostasis (37) and by the present data showing that blockade of NO synthase by L-NAME compromises the gastroprotective activity of ghrelin.
Finally, we have demonstrated that sc administration of 80 µg/kg ghrelin still retains some minor gastroprotective effect, as demonstrated by histology. The peripheral gastroprotective effect of the peptide could involve the beneficial effects on hemodynamic parameters recently documented for ghrelin in both humans (38) and rats (39). However, a central site of action for the gastroprotective effect of sc ghrelin cannot be excluded, because activation of hypothalamic arcuate nucleus was detected when the peptide was administered iv in the rat (40).l, http://www.100md.com
In conclusion, this is the first evidence that ghrelin exerts a potent gastroprotective activity against EtOH-induced lesions, mainly after central administration. The gastroprotective effect of ghrelin is mediated by endogenous NO release and requires the integrity of sensory nerve fibers. In gastroenterological practice, acute hemorrhagic and erosive gastropathy may represent a potentially life-threatening condition (41), often requiring urgent surgery. Ghrelin deserves further attention because it could represent a new interesting pharmacological tool for the treatment of acute erosive gastropathy.
Received July 24, 2002.&&, http://www.100md.com
Accepted for publication October 1, 2002.&&, http://www.100md.com
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